Intercellular communication is an absolute requirement for the coordinated functioning of multi-cellular organisms, and cells have long been known to employ gap junctions and synapses to communicate with their neighbors. A new route of cell-to-cell communication has recently been identified via tunneling membrane nanotubes (TNTs). These are dynamic membrane protrusions, a few hundred nanometers in diameter, that physically link cell bodies over distances of tens of microns and allow for the exchange of cytosolic molecules, membrane components and even organelles between neighboring cells. Transmission of Ca2+ signals along TNTs has been proposed as a means of intercellular communication, which may regulate physiological processes as diverse as gene expression, enzyme activity and electrical excitability. Our modeling studies indicate that passive diffusion of Ca2+ ions along TNTs is inadequate to support efficient transmission of Ca2+ signals between cells. Instead, our observations of local spontaneous and inositol trisphosphate-evoked Ca2+ signals generated within the length of TNTs formed between cultured mammalian cells suggest a mechanism for active propagation of intercellular Ca2+ signals along TNTs. We thus hypothesize that clusters of Ca2+- activated Ca2+ release channels function as amplification sites to overcome limitations of passive diffusion in a chemical analog of electrical transmission of action potentials along axons. Our overall goals are to elucidate the mechanisms underlying this novel mechanism of Ca2+ wave propagation along TNTs, and to explore its role in the physiological and pathological cell-cell communication of Ca2+ signals. Our specific aims are; (1) Using fluorescent calcium indicators we will determine the mechanisms and types of Ca2+ channels involved in regenerative local Ca2+ release events within TNTs, and elucidate the sequestration/buffering systems that shape these localized events in time and space. (2) Utilizing novel superresolution imaging techniques we will map individual Ca2+ release channels with nanometer precision along the TNT and, employing single molecule superresolution imaging, we will explore the diffusional motility of IP3Rs and the contiguous nature of the endoplasmic reticulum along its length. (3) We will explore how local calcium release events coordinate with one another to propagate a Ca2+ wave, the requirements for this process to occur efficiently, and investigate the role for TNTs in spreading aberrant Ca2+ signals in response to cellular stress. Our proposal will provide important mechanistic insights into TNT-mediated propagation of Ca2+ signals and will likely lead to significant advances in our understanding of the physiology and pathophysiological processes involved in this novel mechanism of cell-to-cell communication. PUBLIC HEALTH RELEVANCE: The ability of cells to communicate information between one another is crucial for normal physiology and errors in processing information between cells are known to contribute to diseases such as cancer, Alzheimer's disease and diabetes. Here we propose to study a new form of communication between cells where calcium is used to transmit information directly within very long, thin, tubular extensions of the cell membrane. Our proposal aims to provide important mechanistic insights into how this process occurs and will likely lead to significant advances in our understanding of the physiology and pathophysiological processes involved in cell- to-cell communication.